We present a clever design concept of using femtosecond laser pulses in microscopy by selective excitation or de-excitation of one fluorophore over the other overlapping one. Using either a simple pair of femtosecond pulses with variable delay or using a train of laser pulses at 20-50 Giga-Hertz excitation, we show controlled fluorescence excitation or suppression of one of the fluorophores with respect to the other through wave-packet interference, an effect that prevails even after the fluorophore coherence timescale. Such an approach can be used both under the single-photon excitation as well as in the multi-photon excitation conditions resulting in effective higher spatial resolution. Such high spatial resolution advantage with broadband-pulsed excitation is of immense benefit to multi-photon microscopy and can also be an effective detection scheme for trapped nanoparticles with near-infrared light. Such sub-diffraction limit trapping of nanoparticles is challenging and a two-photon fluorescence diagnostics allows a direct observation of a single nanoparticle in a femtosecond high-repetition rate laser trap, which promises new directions to spectroscopy at the single molecule level in solution. The gigantic peak power of femtosecond laser pulses at high repetition rate, even at low average powers, provide huge instantaneous gradient force that most effectively result in a stable optical trap for spatial control at sub-diffraction limit. Such studies have also enabled us to explore simultaneous control of internal and external degrees of freedom that require coupling of various control parameters to result in spatiotemporal control, which promises to be a versatile tool for the microscopic world.

Recently, digital photography has become an efficient and economic method to assist dermatologists in monitoring skin characteristics. Although this technology has advanced a great deal in resolution and costs, conventional digital cameras continue to only provide qualitative recording of color information. To address this issue, we are developing a compact, quantitative skin imaging camera by employing spatial frequency domain imaging (SFDI), a non-contact approach for determining tissue optical properties over a wide field-of-view. SFDI uses knowledge of optical properties at multiple wavelengths to recover concentrations of tissue constituents such as oxy/deoxy-hemoglobin, water, and melanin. This method has been well researched and presented in laboratory and research settings. The next step in the development of SFDI systems is to make typical systems compact and cheaper using commercial components. We present our findings by performing a component-by-component analysis of key SFDI system components including light sources, projectors, and cameras.

We describe a multispectral continuous-wave diffuse optical tomography (DOT) system that can be used for in vivo three-dimensional (3-D) imaging of seizure dynamics. Fast 3-D data acquisition is realized through a time multiplexing approach based on a parallel lighting configuration - our system can achieve 0.12ms per source per wavelength and up to 14Hz sampling rate for a full set of data for 3-D DOT image reconstruction. The system is validated using both static and dynamic tissue-like phantoms. In vivo rat experiments using both focal and generalized models of seizure are also demonstrated. In the focal seizure experiment, hemodynamic seizure focus was clearly detected and tracked. In the generalized seizure experiment, early hemodynamic responses with heterogeneous patterns were detected several minutes preceding the EEG onset of seizures and widespread hemodynamic changes were found evolving from local regions. Connectivity changes were also found during the development of seizures. This study demonstrates that DOT represents a powerful tool for investigating seizure generation and propagation, elucidating the causes and mechanism of seizures.

Over the last few years, fluorescence imaging for biomedical applications has experienced very rapid growth. An application triggering significant interest is the use of fluorescence for image guidance during surgical interventions. A custom 15x broadband (400-900 nm) macro-zoom objective has been designed, manufactured, and tested for use in image-guided surgery that employs near-infrared (NIR) fluorescence imaging. The lens has been incorporated into the novel FLARE™ imaging system for NIR fluorescence image-guided surgery.

Laparoscope is the essential tool for minimally invasive surgery (MIS) within the abdominal cavity. However, the focal length of a conventional laparoscope is fixed. Therefore, it suffers from the tradeoff between field of view (FOV) and spatial resolution. In order to obtain large optical magnification to see more details, a conventional laparoscope is usually designed with a small working distance, typically less than 50mm. Such a small working distance limits the field of coverage, which causes the situational awareness challenge during the laparoscopic surgery. We developed a multi-resolution foveated laparoscope (MRFL) aiming to address this limitation. The MRFL was designed to support a large working distance range from 80mm to 180mm. It is able to simultaneously provide both wide-angle overview and high-resolution image of the surgical field in real time within a fully integrated system. The high-resolution imaging probe can automatically scan and engage to any subfield of the wide-angle view. During the surgery, MRFL does not need to move; therefore it can reduce the instruments conflicts. The FOV of the wide-angle imaging probe is 80° and that of the high-resolution imaging probe is 26.6°. The maximum resolution is about 45um in the object space at an 80mm working distance, which is about 5 times as good as a conventional laparoscope at a 50mm working distance. The prototype can realize an equivalent 10 million-pixel resolution by using only two HD cameras because of its foveation capability. It saves the bandwidth and improves the frame rate compared to the use of a super resolution camera. It has great potential to aid safety and accuracy of the laparoscopic surgery.

Pinhole is a critical device in single photon confocal microscopy (SPCM) owning to its ability to block the background
noise scattered from back and forth of the focal plane. Without pinhole, the sectioning ability of SPCM will be degraded
and many background noise signals will occurred together with useful signals, and sometimes these bad noises can
submerge the details that we are interested in. However a pinhole with too small diameter will block both background
noises and part of signals and decrease the intensity of the image. Therefore in many cases pinhole size should be
selected carefully. Unfortunately because of constrains in mechanics, a pinhole that can change its size continuously, for
example from 10 μm to 100 μm, is unavailable. For most commercial confocal microscopies, only several discrete
pinhole sizes are provided, such as 10 μm, 30 μm, 60 μm etc. Things will be even harder for some imaging systems
which use the input interface of a single mode fiber as the pinhole of SPCM, and then the pinhole size of these systems
will be fixed, which far limit the optimization of systems’ performance.
In this paper, we design a size-variable pinhole setup that can offer a virtual pinhole with its diameter adjustable, which
includes a physical pinhole (or single mode fiber) and a fine designed zoom relay (ZR) optical system. The
magnification ratio of this ZR can vary smoothly while keeping the conjugation distance unchanged. The aberrations of
the ZR are well balanced and diffraction-limited image performance are obtained so that the virtual pinhole can block
background scattering noise and pass the in-focus signal effectively and accurately. Simulation results are also provided
and discussed.

Point spread function (PSF) phantoms based on unstructured distributions of sub-resolution particles in a transparent matrix have proven effective for evaluating resolution and its spatial variation in optical coherence tomography (OCT) systems. Measurements based on PSF phantoms have the potential to become a standard test method for consistent, objective and quantitative inter-comparison of OCT system performance. Towards this end, we have evaluated three PSF phantoms and investigated their ability to compare the performance of four OCT systems. The phantoms are based on 260-nm-diameter gold nanoshells, submicron-diameter iron oxide particles and 1.5-micron-diameter silica particles. The OCT systems included spectral-domain and swept source systems in free-beam geometries as well as a time-domain system in both free-beam and fiberoptic probe geometries. Results indicated that iron oxide particles and gold nanoshells were most effective for measuring spatial variations in the magnitude and shape of PSFs across the image volume. The intensity of individual particles was also used to evaluate spatial variations in signal intensity uniformity. Significant system-to-system differences in resolution and signal intensity and their spatial variation were readily quantified. The phantoms proved useful for identification and characterization of irregularities such as astigmatism. Particle concentrations of 5000 per cubic millimeter or greater provided accurate determination of performance metrics. Our multi-system inter-comparison provides evidence of the effectiveness of PSF-phantom-based test methods for comparison of OCT system resolution and signal uniformity.

This contribution compares two approaches for the geometric calibration of an optical coherence tomography
(OCT) which forms part of a medical navigation system. For this purpose, a one step and a multi step calibration
is performed with a self-produced 3D reference structure and a high-accurate 6 degrees of freedom (DoF)
parallel robot, respectively. These 3D landmark-based geometric calibrations are based on the identification of
a parameterized grey-box OCT model. We show in experimental results that both methods reduce systematic
errors by more than one order of magnitude.

Spectral variations in contrast enhancement of mucosal vasculature are a key feature of narrow band imaging (NBI) devices. In prior NBI studies, the enhanced visualization of larger, deeper vessels with green light (e.g., 540 nm) relative to violet light (e.g., 415 nm) has often been attributed to the well-known monotonic decrease in scattering coefficient with wavelength in biological tissues. We have developed and implemented numerical and experimental approaches to elucidate and quantify this and other light-tissue interaction effects relevant to NBI. A Monte Carlo model incorporating vessel-like inclusions with a range of diameters (20 to 400 microns) and depths (20 to 400 microns) was used to predict reflectance and fluence distributions in the tissue and calculate vessel contrast values. These results were compared to experimental measurements based on a liquid phantom with a hemoglobin-filled capillary. By comparing results for cases representing mucosa regions with and without blood, we were able to evaluate the relative significance of absorption and scattering on spectral variations in depth-selectivity. Results indicate that at 415 nm, detection of superficial vasculature with NBI was almost entirely dependent on the absorption coefficient of the blood in the vessel of interest. The enhanced visualization of deep vessels at 540 nm bands relative to 415 nm was due primarily to absorption by the superficial vasculature rather than a decrease in scattering coefficient. While computationally intensive, our numerical modeling approach provides unique insights into the light propagation mechanisms underlying this emerging clinical imaging technology.

A micro-lens array based optical detector (MLA-D) has been developed for preclinical in vivo optical imaging applications. While primarily intended for detecting signals from molecular optical probes within living subjects (mice), the MLA-D also can be used effectively to capture the surface of the imaged object in three dimensions from only a few projection angles - a feature that is very important for in vivo optical imaging. In order to study the shape recognition ability of the MLA-D design we have developed a ray-tracing simulation framework. The impact of the following physical MLA-D parameters on surface recognition efficiency can be studied: micro-lens diameter, micro-lens focal length, and sensor pixel size. By using this framework the performance of two surface recognition algorithms - the optical flow method and the multi-projection surface reconstruction (back-projection) method - has been assessed within the specific context of preclinical imaging application. By way of example, the commonly used DigiMouse dataset is adopted to generate simulated raw image data. Results of the simulation framework conform well with the depth-of-field theory, and both surface recognition methods yield comparable, but unsatisfactory results. Whereas the optical flow method reveals the relative shape of the phantom at a comparatively lesser spatial and depth resolution, the back-projection method, while providing higher resolution data, could not resolve concave regions in all cases which needs further investigation. Very promising preliminary results have been attained, however, with the multi-view stereo algorithm that has been implemented most recently.

We developed a novel method for determining the presampling modulation transfer function (MTF) of digital radiography systems from slanted edge images based on Wiener deconvolution. The degraded supersampled edge spread function (ESF) was obtained from simulated slanted edge images with known MTF in the presence of poisson noise, and its corresponding ideal ESF without degration was constructed according to its central edge position. To meet the requirements of the absolute integrable condition of Fourier transformation, the origianl ESFs were mirrored to construct the symmetric pattern of ESFs. Then based on Wiener deconvolution technique, the supersampled line spread function (LSF) could be acquired from the symmetric pattern of degraded supersampled ESFs in the presence of ideal symmetric ESFs and system noise. The MTF is then the normalized magnitude of the Fourier transform of the LSF. The determined MTF showed a strong agreement with the theoritical true MTF when appropriated Wiener parameter was chosen. The effects of Wiener parameter value and the width of square-like wave peak in symmetric ESFs were illustrated and discussed. In conclusion, an accurate and simple method to measure the presampling MTF was established using Wiener deconvolution technique according to slanted edge images.

Efficient light collection is critical in noninvasive deep tissue spectroscopy since only a small fraction of the injected light emerges from any given finite area on the surface of the probed medium. Light collection can be improved by optimizing the contact area between the detection system and the probed medium by means of light guides with large detection areas. Since the form factor of these light guides do not match the entrance of commercial spectrometers, which are usually equipped with a narrow slit to improve their spectral resolution, deep tissue spectrometers are typically custom-built. However, off-the-shelf spectrometers have attractive advantages compared to custom-made units, such as low-cost, small foot-print and availability. In this report, we present simple modifications to an off-the-shelf spectrometer to convert it into a suitable instrument for deep tissue spectroscopy. The modified spectrometer was characterized and compared to a custom-built unit specifically designed for deep tissue spectroscopy. We also present in vivo measurements acquired simultaneously with the two spectrometers in a piglet model of newborn.